Tony Valente

Zac1 is expressed in progenitor/stem cells of the neuroectoderm and mesoderm during embryogenesis: Differential phenotype of the Zac1-expressing cells during development

Zac1, a new zinc‐finger protein that regulates both apoptosis and cell cycle arrest, is abundantly expressed in many neuroepithelia during early brain development. In the present work, we study the expression of Zac1 during early embryogenesis and we determine the cellular phenotype of the Zac1‐expressing cells throughout development. Our results show that Zac1 is expressed in the progenitor/stem cells of several tissues (nervous system, skeleton, and skeletal muscle), because they colocalize with several progenitor/stem markers (Nestin, glial fibrillary acidic protein, FORSE‐1, proliferating cell nuclear antigen, and bromodeoxyuridine). In postnatal development, Zac1 is expressed in all phases of the life cycle of the chondrocytes (from proliferation to apoptosis), in some limbic γ‐aminobutyric acid‐ergic neuronal subpopulations, and during developmental myofibers. Therefore, the intense expression of Zac1 in the progenitor/stem cells of different cellular lineages during the proliferative cycle, before differentiation into postmitotic cells, suggests that Zac1 plays an important role in the control of cell fate during neurogenesis, chondrogenesis, and myogenesis. Developmental Dynamics 233:667–679, 2005.

Expression pattern of Zac1 mouse gene, a new zinc-finger protein that regulates apoptosis and cellular cycle arrest, in both adult brain and along development.

Using in situ hybridization, we analyzed the expression pattern of the Zac1 gene in mouse brain during the embryonic and postnatal development. Zac1 is a new gene that regulates extensive apoptosis and cell cycle arrest through unrelated pathways. At embryonic stages, strong expression was observed in brain areas with active proliferation (ventricular zone and numerous neuroepithelius) and in nervous system (neural retina and neural tube). In addition, some areas with differentiation activity were noticeably labeled such as arcuate nucleus and amygdaloid region of the brain together with other embryonic sites (hindlimb, forelimb and somites). From P0 onwards, the expression appeared in some proliferative areas, such as subventricular zone and cerebellum (external granular layer and Purkinje cells) and in some synaptic plasticity areas, such as the dorso and ventromedial hypothalamic nuclei, arcuate nucleus, ventral thalamic nucleus.

Postnatal development of zinc-rich terminal fields in the brain of the rat.

The appearance and distribution of zinc-rich terminal fields in the rat forebrain was analyzed at 12 stages of postnatal development using the selenium method. Zinc stain was detected in neonates in piriform, cingulate, and motor cortices, septal area, and hippocampal formation. In the neocortex, a laminar pattern appeared progressively following an inside-out gradient: layer VI at postnatal day 0 (P0), layer V at P1, layers Va and Vb at P5, layer II-III at P9, and layer IV at P12. In the hippocampal formation the layered pattern in the dentate molecular layer appeared at P1-P3, and in the hilus and mossy fibers the stain was observed at P5. Patches in the caudate-putamen were sharply delimited at P1-P3. At these ages, staining was observed in the amygdaloid complex. In the thalamic and hypothalamic nuclei, stain appeared at P5-P7. Thus, a general increase in vesicular zinc over different telencephalic areas was determined until P15-P21, which was followed by a slight decrease thereafter (at P41). The increased stain in zinc-rich terminal fields is consistent with the development of telencephalic circuits. The rise in zinc might be relevant for the establishment and maturation of these circuits. On the other hand, the decrease in staining for zinc at later stages might be due to methodological problems but it might also reflect pruning of supernumerary connections and programmed cell death affecting zinc-rich circuits.

Correlates of cerebrospinal fluid levels of oligomeric- and total-α-synuclein in premotor, motor and dementia stages of Parkinson’s disease

High-oligomeric and low-total-α-synuclein cerebrospinal fluid (CSF) levels have been found in Parkinson’s disease (PD), but with inconsistent or limited data, particularly on their clinical and structural correlates in earliest (premotor) or latest (dementia) PD stages. We determined CSF oligomeric- and total-α-synuclein in 77 subjects: 23 with idiopathic REM-sleep behaviour disorder (iRBD, a condition likely to include a remarkable proportion of subjects in the premotor stage of PD) and 41 with PD [21 non-demented (PDND)xa0+xa020 demented (PDD)], intended to reflect the premotor–motor–dementia PD continuum, along with 13 healthy controls. The study protocol also included the Unified PD Rating Scale motor-section (UPDRS-III), mini mental state examination (MMSE), neuropsychological cognitive testing, 3T brain MRI for cortical-thickness analyses, CSF τ and CSF Aβ. CSF oligomeric-α-synuclein was higher in PDND than iRBD and in PDD than iRBD and controls, and correlated with UPDRS-III, MMSE, semantic fluency and visuo-perceptive scores across the proposed premotor–motor–dementia PD continuum (iRBDxa0+xa0PDNDxa0+xa0PDD). CSF total-α-synuclein positively correlated with age, CSF Aβ, and, particularly, CSF τ, tending towards lower levels in PD (but not iRBD) vs. controls only when controlling for CSF τ. Low CSF total-α-synuclein was associated with dysfunction in phonetic-fluency (a frontal-lobe function) in PD and with frontal cortical thinning in iRBD and PDND independently of CSF τ. Conversely, the associations of high (instead of low)xa0CSF total-α-synuclein with posterior-cortical neuropsychological deficits in PD and with posterior cortical thinning in PDD were driven by high CSF τ. These findings suggest that CSF oligomeric- and total-α-synuclein have different clinical, neuropsychological and MRI correlates across the proposed premotor–motor–dementia PD continuum. CSF total-α-synuclein correlations with CSF τ and Aβ support the hypothesis of an interaction among these proteins in PD, with CSF τ probably influencing the presence of high (instead of low)xa0CSF total-α-synuclein and its correlates mostly in the setting of PD-related dementia.

Developmental Expression of ZnT3 in Mouse Brain: Correlation between the Vesicular Zinc Transporter Protein and Chelatable Vesicular Zinc (CVZ) Cells. Glial and Neuronal CVZ Cells Interact

We examine the expression pattern of ZnT3 in the cerebral and cerebellar areas of mouse brain throughout development. During embryogenesis and early postnatal stages, ZnT3 transcripts were detected in several areas. Label was clear in areas related to proliferation and differentiation. As development proceeded, the label gradually disappeared in these areas and increased in the chelatable vesicular zinc (CVZ) system. To assess whether ZnT3 was expressed in all CVZ cells, its distribution pattern was studied through postnatal stages using a retrograde zinc transport method. While the ZnT3 expression pattern and the distribution of CVZ cells coincided from P12 to adulthood, this coincidence was not detected in early postnatal days. Moreover, immunohistochemical procedures highlighted a differential phenotype within CVZ cells throughout postnatal development. These findings suggest the presence of different CVZ cell subpopulations throughout brain development and, consequently, the existence of distinct chelatable vesicular zinc pools.

CD200R1 and CD200 expression are regulated by PPAR-γ in activated glial cells.

The mechanisms that control microglial activation are of interest, since neuroinflammation, which involves reactive microglia, may be an additional target in the search for therapeutic strategies to treat neurodegenerative diseases. Neuron‐microglia interaction through contact‐dependent or independent mechanisms is involved in the regulation of the microglial phenotype in both physiological and pathological conditions. The interaction between CD200, which is mainly present in neurons but also in astrocytes, and CD200R1, which is mainly present in microglia, is one of the mechanisms involved in keeping the microglial proinflammatory phenotype under control in physiological conditions. Alterations in the expression of CD200 and CD200R1 have been described in neurodegenerative diseases, but little is known about the mechanism of regulation of these proteins under physiological or pathological conditions. The aim of this work was to study the modulation of CD200 and CD200R1 expression by peroxisome proliferator‐activated receptor gamma (PPAR‐γ), a transcription factor involved in the control of the inflammatory response. Mouse primary neuronal and glial cultures and neuron‐microglia cocultures were treated with the PPAR‐γ endogenous ligand 15‐deoxy‐Δ12, 14‐prostaglandin J2 (15d‐PGJ2) in the presence and absence of lipopolysaccharide plus interferon‐γ (LPS/IFN‐γ)‐induced glial activation. We show that 15d‐PGJ2 inhibits the pro‐inflammatory response and prevents both CD200R1 downregulation and CD200 upregulation in reactive glial cells. In addition, 15d‐PGJ2 abrogates reactive‐microglia induced neurotoxicity in neuron‐microglia cultures through a CD200‐CD200R1 dependent mechanism. These results suggest that PPAR‐γ modulates CD200 and CD200R1 gene expression and that CD200‐CD200R1 interaction is involved in the anti‐inflammatory and neuroprotective action of PPAR‐γ agonists. GLIA 2014;62:982–998

C/EBPβ expression in activated microglia in amyotrophic lateral sclerosis.

Neuroinflammation is thought to play a pathogenic role in many neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). In this study we demonstrate that the expression of nitric oxide (NO) synthase-2 (NOS2), and cyclooxygenase (COX)-2 induced by lipopolysaccharide (LPS) with interferon-γ is higher in microglial-enriched cultures from G93A-SOD1 mice, an ALS animal model, than from wild type mice. The levels of CCAAT/enhancer binding protein β (C/EBPβ), a transcription factor that regulates proinflammatory gene expression, are also upregulated in activated G93A-SOD1 microglial cells. In vivo, systemic lipopolysaccharide also induces an exacerbated neuroinflammatory response in G93A-SOD1 mice versus wild type mice, with increased expression of glial fibrillary acidic protein (GFAP), CD11b, nitric oxide synthase-2, cyclooxygenase-2, proinflammatory cytokines, and C/EBPβ. Finally, we report that C/EBPβ is expressed by microglia in the spinal cord of ALS patients. This is the first demonstration to our knowledge of microglial C/EBPβ expression in human disease. Altogether these findings indicate that G93A-SOD1 expression results in an exacerbated pattern of neuroinflammation and suggest that C/EBPβ is a candidate to regulate the expression of potentially neurotoxic genes in microglial cells in ALS.

CCAAT/enhancer binding protein δ regulates glial proinflammatory gene expression

The transcription factor CCAAT/enhancer binding protein δ (C/EBPδ) is expressed in activated astrocytes and microglia and can regulate the expression of potentially detrimental proinflammatory genes. Thexa0objective of this study was to determine the role of C/EBPδ in glial activation. To this end, glial activation was analyzed in primary glial cultures and in the central nervous system from wild type and C/EBPδ(-/-) mice. Inxa0vitro studies showed that the expression of proinflammatory genes nitric oxide (NO)synthase-2, cyclooxygenase-2, and interleukin (IL)-6 in glial cultures, and the neurotoxicity elicited by microglia in neuron-microglia cocultures, were decreased in the absence of C/EBPδ when cultures were treated with lipopolysaccharide (LPS) and interferon γ, but not with LPS alone. In C/EBPδ(-/-) mice, systemic LPS-induced brain expression of NO synthase-2, tumor necrosis factor-α, IL-1β, and IL-6 was attenuated. Finally, increased C/EBPδ nuclear expression was observed in microglial cells from amyotrophic lateral sclerosis patients and G93A-SOD1 mice spinal cord. These results demonstrate that C/EBPδ plays a key role in the regulation of proinflammatory gene expression in glial activation and suggest that C/EBPδ inhibition has potential for the treatment of neurodegenerative disorders, in particular, amyotrophic lateral sclerosis.

CCAAT/enhancer binding protein β regulates prostaglandin E synthase expression and prostaglandin E2 production in activated microglial cells.

The eicosanoid prostaglandin E2 (PGE2) plays important roles in neuroinflammation and it is produced by the sequential action of the enzymes cyclooxygenase‐2 (COX‐2) and prostaglandin E synthase (PTGES). The expression of both enzymes and the production of PGE2 are increased in neuroinflammation. The objective of this study was to elucidate whether the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) regulates the expression of prostaglandin synthesis enzymes in neuroinflammation. To this aim, the expression of these enzymes in wild‐type and C/EBPβ‐null mice was analyzed in vitro and in vivo. In mixed glial cultures, lipopolysaccharide (LPS)u2009±u2009interferon γ (IFN‐γ) induced C/EBPβ binding to COX‐2 and PTGES promoters. LPSu2009±u2009IFN‐γ‐induced increases in PTGES expression and in PGE2 production in mixed glial and microglial cultures were abrogated in the absence of C/EBPβ. Also, increased brain PTGES expression induced by systemic LPS administration was markedly reduced in C/EBPβ‐null mice. In contrast to PTGES, the induction of COX‐2 expression in vitro or in vivo was not markedly affected by the absence of C/EBPβ. These results demonstrate that C/EBPβ regulates PTGES expression and PGE2 production by activated microglial cells in vitro and point to C/EBPβ as a regulator of PTGES expression in vivo in the inflamed central nervous system. Altogether, these findings strengthen the proposed role of C/EBPβ as a key player in the orchestration of neuroinflammatory gene response. GLIA 2013;61:1607–1619

Immunohistochemical study of semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 in the hippocampal vasculature: Pathological synergy of Alzheimer's disease and diabetes mellitus

Semicarbazide‐sensitive amine oxidase/vascular adhesion protein‐1 (SSAO/VAP‐1) is involved in vascular endothelial damage as well as in the vascular degeneration underlying diabetes mellitus and Alzheimers disease (AD). Recent evidence suggests that classic pathological features of AD are more pronounced in diabetic mellitus patients. To investigate the expression and distribution of SSAO/VAP‐1 in the two pathologies, we have performed an immunohistochemical study in human hippocampal vessels of AD, AD with diabetic mellitus (ADD), diabetic mellitus (DM), and nondemented (ND) patients. The present results demonstrate major vessel accumulation of both SSAO/VAP‐1 and amyloid‐β immunolabeling intensity in ADD compared with AD patients. Interestingly, nearly damaged vessels with high levels of SSAO/VAP‐1 also showed increased oxidative damage markers (AGE, RAGE, and SOD‐1) and glial activation (GFAP and HLA). Overall, this work suggests that high vascular SSAO/VAP‐1 levels in human hippocampus may contribute to vascular degeneration, which can explain the severe progression in patients with both pathologies.